Diseases caused by bacterial infections have significant morbidity and mortality in man and other mammals. The infection process consists of three stages: bacterial entry and colonization of the Host; bacterial invasion and growth in host tissues along with the appearance of toxic substances; and the host response.
Bacterial infections can be classed broadly into those caused by Gram positive bacteria, such as the Staphylococci and Streptococci, and those caused by Gram negative bacteria, such as Escherichia coli. Gram positive bacteria have a typical lipid bilayer cytoplasmic membrane surrounded by a rigid cell wall. The cell wall is composed mainly of peptidoglycan, a polymer of N-acetylglucosamine and N-acetyl muramic acid crosslinked by a peptide comprising alternating D- and L-amino acids. In addition, the outer cell wall of Gram-positive bacteria comprises a complex of polysaccharides, proteins, teichoic acids, and lipoteichoic acids. By contrast, Gram-negative bacteria have a much smaller peptidoglycan layer, an outer membrane that contains lipopolysaccharide which lacks the complex layer of carbohydrate and teichoic acids.
Antibiotics are substances produced by various species of microorganisms (bacteria, fungi) that suppress the growth of other microorganisms and may eventually destroy them. In addition, common usage extends the term antibiotic to include antibacterial agents which are semi-synthetic antibiotics, i.e. chemically modified bacterial antibiotics, as well as synthetic antibacterial agents (e.g. sulphonamides) which are not products of microbes. Also included in the term “antibiotic” are various peptides found in host defence systems which are produced locally in response to colonisation by or invasion of microorganisms (e.g. peptides produced by amphibians, including the peptide magainin). Hundreds of antibiotics have been identified, and many have been developed to the stage where they are of value in the therapy of infectious diseases.
Several schemes have been proposed to classify and group antimicrobial agents. The most common classification has been based on chemical structure and proposed mechanism of action, as follows: (1) agents that act directly on the cell membrane of the microorganism, affecting permeability and leading to leakage of the intracellular compounds, such as detergents, cationic peptides, gramicidin A, and pplymyxin; (2) agents that inhibit synthesis of bacterial cell walls and includes the beta-lactams, cephalasporins and glycopeptides; (3) agents that affect bacterial protein synthesis including tetracycline and chloramphenicol; (4) agents that act as antimetabolites and interfere with the bacterial synthesis of folic acid, such as the sulphonamides; and (5) agents that inhibit nucleic acid synthesis or activity such as quinolones.
Peptide anti-bacterial agents which act directly on the bacterial membrane cause a general permeabilisation or modification of the bacterial cytoplasmic membrane. This results from the binding of peptides to components of the outer membrane surface, causing reorganisation of membrane structure and the creation of pores through which the intracellular contents may leak. Generally, these features are associated with an amphiphilic peptide nature often including helical secondary structure and a net positive charge. Peptide antibiotics having this mode of action include the magainins, defensins, and the lantibiotics such as nisin. The activity of, this class of antibiotics is directed towards bacteria rather than mammalian cells because the positive charged residues of the antibiotic interact with negatively charged lipids which are found predominantly in bacterial rather than mammalian cell membranes.
In particular, the magainins are a class of amphiphilic α-helical peptides found in the skin of the African clawed frog (Xenopus laevis). Peptides of this class (which also include bombinin from amphibians [Gibson, B. W., Tang, D., Mandrell, R., Kelly, M. & Spindel, E. R. (1991) J. Biol. Chem. 266, 223103–23111], melittin from bee venom [Habermann, E. (1972) Science, 177, 314–322], and alamethicin from fungi [Latorre, R. & Alvarez, S. (1981) Physiol. Rev., 61, 77–150]) cause disruption of membrane potential at low concentrations, and membrane lysis via insertion at higher concentrations.
One group of antibiotics that has received widespread attention due to their clinical efficacy is the glycopeptide group of antibiotics. These agents consist of a rigid, cyclised heptapeptide backbone which may be substituted with a variety of amino and non-amino sugars. The amino sugar moieties of some members of this class contain N-acyl, N-alkyl, or N-aryl substitutions. Two antibiotics in this class are vancomycin and teicoplanin. Vancomycin is produced by Streptococcus orientalis, an actinomycete isolated from soil samples in Indonesia and India. The antibiotic was purified and its properties described shortly after its discovery (McCormick et al., 1956). Vancomycin is a complex tricyclic glycopeptide with a molecular mass of approximately 1500 Da. Its structure was determined by X-ray analysis (Sheldrick et al., 1978):

Vancomycin is active primarily against Gram-positive bacteria. Strains of bacteria are considered susceptible at a minimum inhibitory concentration of less than or equal to 4 μg/mL. Strep. Pyogenes, Strep. Pneumoniae, Corynebacteraium spp. are highly susceptible, as are most strains of Enterococcus spp. Most species of Actinomyces and Clostridium spp. are also sensitive to vancomycin, but at higher concentrations of antibiotic. Vancomycin is employed only to treat serious infections and is particularly useful in the management of infections due to methicillin-resistant staphylococci, including pneumonia, emphysema, endocarditis, osteomyelitis, and soft-tissue abscesses. The agent is also extremely useful in the treatment of staphylococcal infections in patients who are allergic to penicillins and cephalosporins.
Vancomycin inhibits the synthesis of the cell wall in sensitive bacteria by blocking the cross-linking of the sugar and peptidic components of peptidoglycans during the synthesis of the bacterial cell wall. Without sufficient cross-linking, the cell wall becomes mechanically fragile and the bacteria lyse when subjected to changes in osmotic pressure. Vancomycin binds with high affinity to the D-alanyl-D-alanine (D-Ala-D-Ala) terminus of the pentapeptide portion of the peptidoglycan precursor before cross-linking. The D-Ala-D-Ala dipeptide forms complementary hydrogen bonds with the peptide backbone of vancomycin. It is thought that the vancomycin-peptidoglycan complex physically blocks the action of the transpeptidase enzyme and thereby inhibits the formation of the peptide cross-bridges that strengthens the peptidoglycan. This activity also leads to the accumulation of peptidoglycan precursors in the bacterial cytoplasm.
Resistance to antibiotics is well documented and the resistant strains are a potential major threat to the wellbeing of mankind. Bacteria become resistant to an antimicrobial agent because either the drug fails to reach its target; the drug is inactivated, or because the target is altered. For example, some bacteria produce enzymes that reside in or within the cell surface and inactivate the drug, while others possess impermeable cell membranes that prevent influx of the drug.
Several types of resistance have been described for vancomycin, including the VanA-C types. The VanA phenotype is inducible by vancomycin and confers resistance to both teicoplanin and vancomycin. The VanA phenotype is mediated by the transposable element Tn1546 or closely related elements (Arthur et al., 1993). The transposon encodes a dehydrogenase (VanH) that reduces pyruvate to D-lactate (D-lac), and a ligase of broad substrate specificity (VanA) that catalyses the formation of an ester bond between D-Ala and D-Lac (Dukta-Malen et al., 1990; Bugg et al., 1991). The resulting D-Ala-D-Lac depsipeptide replaces the dipeptide D-Ala-D-Ala in the pathway of peptidoglycan synthesis. The substitution eliminates a hydrogen bond that is critical for antibiotic binding (Bugg et al., 1991). The VanB phenotype is also induced upon exposure to vancomycin; however, in contrast to the VanA phenotype, these microorganisms are not resistant to teicoplanin because teicoplanin does not induce the expression of the genes required for resistance in VanB bacteria (Arthur et al., 1996; Evers and Courvalin, 1996). Resistance to vancomycin by bacteria of the VanB phenotype occurs through a similar mechanism to VanA resistance, namely the substitution of the terminal D-Ala-A-Ala peptidoglycan precursor on the immature peptidoglycan by the D-Ala-D-Lac depsipeptide. One further vancomycin-resistant phenotype has been described (VanC) in enterococci belonging to the species E. gallinarum, E. casseliflavus and E. flavescens. These bacteria are intrinsically resistant to low levels of vancomycin and are susceptible to teicoplanin. Resistance results from the production of peptidoglycan precursors ending in D-Serine (Billot-Klein et al., 1994; Reynolds et al., 1994).
Substitution of D-Ala by D-Ser at the carboxy-terminus of the peptidoglycan precursor analogues lowers the affinity of the precursors for vancomycin with a relatively small change in the affinity for teicoplanin (Billot-Klein et al., 1994). The emergence and dissemination of high-level resistance to glycopeptides in enterococci in the past decade has resulted in clinical isolates resistant to all antibiotics of proven efficacy (Handwerger et al., 1992; Handwerger et al., 1993). The incidence of glycopeptide resistance among clinical isolates is increasing and enterococci have become important as nosocomial pathogens and as a reservoir of resistance genes (Murray 1990; Woodford et al., 1995). Nosocomial infection with multidrug resistant strains is potentially catastrophic and there is a need to identify novel anti-bacterial agents or methods of controlling bacterial infections.
Approaches that have been used to combat the emergence of antibiotic resistant strains include the modification of existing antibiotics to improve their potency against resistant organisms, or the discovery of new peptide antibiotics which kill their targets by permeabilizing the bacterial plasma membrane. Examples of the first approach have recently focussed on creating derivatives of glycopeptides such as vancomycin.
Functionalisation of the carboxyl terminal of vancomycin using the coupling agent 2-(1-hydroxybenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) has been successful in attaching short peptide sequences, both in solution and solid phases [Sundram, U. N. and Griffin, J. H. (1995) J. Org. Chem. 60 1102–1103]. The aminosugar and terminal amine moieties of vancomycin and related antibiotics. have also been derivatised. In a reductive alkylation approach, a series of compounds alkylated on the vancosamine sugar was created, some of which showed greatly improved activity vs vancomycin resistant bacterial strains [Cooper, R. D. G. et al. (1996) J. Antibiotics 49, 575–581; Rodriguez, M. J. et al. (1998) J. Antibiotics 51, 560–569].
WO-A-98/02454 describes polypeptide derivatives in which a soluble therapeutic polypeptide is modified with an entity of general structure:-(L-[W])n-X  (I)in which each L is independently a flexible linker group, each W is independently a peptidic membrane-binding element, n is an integer greater than or equal to one, and X is a peptidic or non-peptidic membrane-binding or insertive element.
Structures of type (I) represent a combinatorial array of membrane-interactive elements whose attachment to soluble polypeptides was found to mediate binding of those polypeptides to the outer cell membrane of mammalian cells. This gave rise to therapeutic benefits, particularly in the case of regulators of complement activation acting as cytoprotectants and anti-inflammatory agents (e.g. Dong, J. et al, (1999) Mol. Immunol. 36 957–963).